The hypoxia-inducible factor EPAS1 is required for spermatogonial stem cell function in regenerative conditions.

In this study we explored the role of hypoxia and the hypoxia-inducible transcription factor EPAS1 in regulating spermatogonial stem cell (SSC) function in the mouse testis. We have demonstrated that SSCs reside in hypoxic microenvironments in the testis through utilization of the oxygen-sensing probe pimonidazole, and by confirming the stable presence of EPAS1, which is degraded at >5% O2. Through the generation of a germline-specific Epas1 knockout mouse line, and through modulation of EPAS1 levels in primary cultures of spermatogonia with the small drug molecule Daprodustat, we have demonstrated that EPAS1 is required for robust SSC function in regenerative conditions (post-transplantation and post-chemotherapy), via the regulation of key cellular processes such as metabolism. These findings shed light on the relationship between hypoxia and male fertility and will potentially facilitate optimization of in vitro culture conditions for infertility treatment pipelines using SSCs, such as those directed at pediatric cancer survivors.

Introduction: The Why's and How's for Studying Spermatogenesis and Spermatogonial Stem Cells.

Spermatogenesis is maintained throughout adulthood by a pool of adult stem cells termed spermatogonial stem cells (SSCs). Research investigations into spermatogenesis can provide insight into the etiology of certain types of male infertility (e.g., Sertoli cell only syndrome), elucidate means of improving food animal production, reveal new therapeutic avenues to address naturally occurring defects in sperm production, mitigate iatrogenic male infertility (e.g., arising from cancer therapy), and potentially intervene for male contraception. This chapter will serve as a commentary about why studying spermatogenesis is important, including a high-level overview of spermatogonia and SSCs, and make the case for a critical need for use of stringent definitions for SSCs and experimental platforms that allow for clear distinction of the multiple types of spermatogonia that exist in testes of mammals.

Culture of Kenyan Goat (Capra hircus) Undifferentiated Spermatogonia in Feeder-Free Conditions.

The undifferentiated spermatogonial population in mammalian testes contains a spermatogonial stem cell (SSC) population that can regenerate continual spermatogenesis following transplantation. This capacity has the potential to be exploited as a surrogate sires breeding tool to achieve widespread dissemination of desirable genetics in livestock production. Because SSCs are relatively rare in testicular tissue, the ability to expand a population in vitro would be advantageous to provide large numbers for transplantation into surrogate recipient males. Here, we evaluated conditions that would support long-term in-vitro maintenance of undifferentiated spermatogonia from a goat breed that is endemic to Kenyan livestock production. Single-cell suspensions enriched for undifferentiated spermatogonia from pre-pubertal bucks were seeded on laminin-coated tissue culture plates and maintained in a commercial media based on serum-free composition. The serum-free media was conditioned on goat fetal fibroblasts and supplemented with a growth factor cocktail that included glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), stromal cell-derived factor (SDF), and fibroblast growth factor (FGF) before use. Over 45 days, the primary cultures developed a cluster morphology indicative of in-vitro grown undifferentiated spermatogonia from other species and expressed the germ cell marker VASA, as well as the previously defined spermatogonial marker such as promyelocytic leukemia zinc finger (PLZF). Taken together, these findings provide a methodology for isolating the SSC containing undifferentiated spermatogonial population from goat testes and long-term maintenance in defined culture conditions.

A regulatory role for CHD4 in maintenance of the spermatogonial stem cell pool.

Maintenance and self-renewal of the spermatogonial stem cell (SSC) population is the cornerstone of male fertility. Here, we have identified a key role for the nucleosome remodeling protein CHD4 in regulating SSC function. Gene expression analyses revealed that CHD4 expression is highly enriched in the SSC population in the mouse testis. Using spermatogonial transplantation techniques it was established that loss of Chd4 expression significantly impairs SSC regenerative capacity, causing a ∼50% reduction in colonization of recipient testes. An scRNA-seq comparison revealed reduced expression of "self-renewal" genes following Chd4 knockdown, along with increased expression of signature progenitor genes. Co-immunoprecipitation analyses demonstrated that CHD4 regulates gene expression in spermatogonia not only through its traditional association with the remodeling complex NuRD, but also via interaction with the GDNF-responsive transcription factor SALL4. Cumulatively, the results of this study depict a previously unappreciated role for CHD4 in controlling fate decisions in the spermatogonial pool.

Proper timing of a quiescence period in precursor prospermatogonia is required for stem cell pool establishment in the male germline.

The stem cell-containing undifferentiated spermatogonial population in mammals, which ensures continual sperm production, arises during development from prospermatogonial precursors. Although a period of quiescence is known to occur in prospermatogonia prior to postnatal spermatogonial transition, the importance of this has not been defined. Here, using mouse models with conditional knockout of the master cell cycle regulator Rb1 to disrupt normal timing of the quiescence period, we found that failure to initiate mitotic arrest during fetal development leads to prospermatogonial apoptosis and germline ablation. Outcomes of single-cell RNA-sequencing analysis indicate that oxidative phosphorylation activity and inhibition of meiotic initiation are disrupted in prospermatogonia that fail to enter quiescence on a normal timeline. Taken together, these findings suggest that key layers of programming are laid down during the quiescent period in prospermatogonia to ensure proper fate specification and fitness in postnatal life.

Spermatogonial Stem Cell Numbers Are Reduced by Transient Inhibition of GDNF Signaling but Restored by Self-Renewing Replication when Signaling Resumes.

One cause of human male infertility is a scarcity of spermatogonial stem cells (SSCs) in testes with Sertoli cells that neither produce adequate amounts of GDNF nor form the Sertoli-Sertoli junctions that form the blood-testis barrier (BTB). These patients raise the issue of whether a pool of SSCs, depleted due to inadequate GDNF stimulation, will expand if normal signaling is restored. Here, we reduce adult mouse SSC numbers by 90% using a chemical-genetic approach that reversibly inhibits GDNF signaling. Signal resumption causes all remaining SSCs to replicate immediately, but they primarily form differentiating progenitor spermatogonia. Subsequently, self-renewing replication restores SSC numbers. Testicular GDNF levels are not increased during restoration. However, SSC replication decreases as numbers of SSCs and progenitors increase, suggesting important regulatory interactions among these cells. Finally, sequential loss of SSCs and then pachytene spermatocytes causes dissolution of the BTB, thereby recapitulating another important characteristic of some infertile men.

Donor-derived spermatogenesis following stem cell transplantation in sterile NANOS2 knockout males.

Spermatogonial stem cell transplantation (SSCT) is an experimental technique for transfer of germline between donor and recipient males that could be used as a tool for biomedical research, preservation of endangered species, and dissemination of desirable genetics in food animal populations. To fully realize these potentials, recipient males must be devoid of endogenous germline but possess normal testicular architecture and somatic cell function capable of supporting allogeneic donor stem cell engraftment and regeneration of spermatogenesis. Here we show that male mice, pigs, goats, and cattle harboring knockout alleles of the NANOS2 gene generated by CRISPR-Cas9 editing have testes that are germline ablated but otherwise structurally normal. In adult pigs and goats, SSCT with allogeneic donor stem cells led to sustained donor-derived spermatogenesis. With prepubertal mice, allogeneic SSCT resulted in attainment of natural fertility. Collectively, these advancements represent a major step toward realizing the enormous potential of surrogate sires as a tool for dissemination and regeneration of germplasm in all mammalian species.

Translational Repression of G3BP in Cancer and Germ Cells Suppresses Stress Granules and Enhances Stress Tolerance.

Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.

Isolation of canine adipose-derived mesenchymal stem cells from falciform tissue obtained via laparoscopic morcellation: A pilot study.

OBJECTIVE: To evaluate the feasibility of stem cell isolation from falciform fat harvested via laparoscopic morcellation. STUDY DESIGN: Pilot study. ANIMALS: Eleven client-owned dogs. METHODS: Falciform was harvested traditionally via laparotomy and laparoscopically via tissue morcellation. Harvested tissue was processed with a commercially available adipose tissue dissociation kit to obtain a stromal vascular fraction (SVF). Cells were subsequently labeled for CD90, CD45, and CD44 cell surface antigens by using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting flow cytometry. CD90+ cells were quantitated, and their viability was assessed with a hemocytometer and a trypan blue exclusion test of cell viability. RESULTS: No perioperative complications occurred in dogs undergoing laparoscopic morcellation. Laparoscopically and traditionally harvested samples yielded an average of 0.39 (±0.1) × 106 and 0.33 (±0.1) × 106 CD90+ cells, respectively, per 10 million SVF cells. CD90+ cell viability after MACS was 89% (±11%) for morcellated and 86% (±7%) for traditionally harvested samples. Neither CD90+ cell quantity nor viability was different between samples obtained via traditional laparotomy vs laparoscopic morcellation (P = .38 and P = .63, respectively). Populations of CD90+ cells isolated with each harvest technique had similar CD44 and CD45 expression profiles. CONCLUSION: Viable populations of CD90+ cells with similar CD44/CD45 expression profiles were isolated from laparoscopically morcellated and traditionally harvested falciform tissue. No appreciable morbidity was associated with laparoscopic falciform morcellation. CLINICAL SIGNIFICANCE: Laparoscopic morcellation is a safe and effective minimally invasive approach to falciform tissue harvest for adipose-derived mesenchymal stem cell isolation.

Developmental kinetics and transcriptome dynamics of stem cell specification in the spermatogenic lineage.

Continuity, robustness, and regeneration of cell lineages relies on stem cell pools that are established during development. For the mammalian spermatogenic lineage, a foundational spermatogonial stem cell (SSC) pool arises from prospermatogonial precursors during neonatal life via mechanisms that remain undefined. Here, we mapped the kinetics of this process in vivo using a multi-transgenic reporter mouse model, in silico with single-cell RNA sequencing, and functionally with transplantation analyses to define the SSC trajectory from prospermatogonia. Outcomes revealed that a heterogeneous prospermatogonial population undergoes dynamic changes during late fetal and neonatal development. Differential transcriptome profiles predicted divergent developmental trajectories from fetal prospermatogonia to descendant postnatal spermatogonia. Furthermore, transplantation analyses demonstrated that a defined subset of fetal prospermatogonia is fated to function as SSCs. Collectively, these findings suggest that SSC fate is preprogrammed within a subset of fetal prospermatogonia prior to building of the foundational pool during early neonatal development.

MAGE cancer-testis antigens protect the mammalian germline under environmental stress.

Ensuring robust gamete production even in the face of environmental stress is of utmost importance for species survival, especially in mammals that have low reproductive rates. Here, we describe a family of genes called melanoma antigens (MAGEs) that evolved in eutherian mammals and are normally restricted to expression in the testis (http://MAGE.stjude.org) but are often aberrantly activated in cancer. Depletion of Mage-a genes disrupts spermatogonial stem cell maintenance and impairs repopulation efficiency in vivo. Exposure of Mage-a knockout mice to genotoxic stress or long-term starvation that mimics famine in nature causes defects in spermatogenesis, decreased testis weights, diminished sperm production, and reduced fertility. Last, human MAGE-As are activated in many cancers where they promote fuel switching and growth of cells. These results suggest that mammalian-specific MAGE genes have evolved to protect the male germline against environmental stress, ensure reproductive success under non-optimal conditions, and are hijacked by cancer cells.

Spermatogonial Stem Cell Transplantation: Insights and Outlook for Domestic Animals.

The demand for food will increase to an unprecedented level over the next 30 years owing to human population expansion, thus necessitating an evolution that improves the efficiency of livestock production. Genetic gain to improve production traits of domestic animal populations is most effectively achieved via selective use of gametes from animals deemed to be elite, and this principle has been the basis of selective breeding strategies employed by humans for thousands of years. In modern-day animal agriculture, artificial insemination (AI) has been the staple of selective breeding programs, but it has inherent limitations for applications in beef cattle and pig production systems. In this review, we discuss the potential and current state of development for a concept termed Surrogate Sires as a next-generation breeding tool in livestock production. The scheme capitalizes on the capacity of spermatogonial stem cells to regenerate sperm production after isolation from donor testicular tissue and transfer into the testes of a recipient male that lacks endogenous germline, thereby allowing the surrogate male to produce offspring with the donor haplotype via natural mating. This concept provides an effective selective breeding tool to achieve genetic gain that is conducive for livestock production systems in which AI is difficult to implement.

The Mammalian Spermatogenesis Single-Cell Transcriptome, from Spermatogonial Stem Cells to Spermatids.

Spermatogenesis is a complex and dynamic cellular differentiation process critical to male reproduction and sustained by spermatogonial stem cells (SSCs). Although patterns of gene expression have been described for aggregates of certain spermatogenic cell types, the full continuum of gene expression patterns underlying ongoing spermatogenesis in steady state was previously unclear. Here, we catalog single-cell transcriptomes for >62,000 individual spermatogenic cells from immature (postnatal day 6) and adult male mice and adult men. This allowed us to resolve SSC and progenitor spermatogonia, elucidate the full range of gene expression changes during male meiosis and spermiogenesis, and derive unique gene expression signatures for multiple mouse and human spermatogenic cell types and/or subtypes. These transcriptome datasets provide an information-rich resource for studies of SSCs, male meiosis, testicular cancer, male infertility, or contraceptive development, as well as a gene expression roadmap to be emulated in efforts to achieve spermatogenesis in vitro.

Functional assessment of spermatogonial stem cell purity in experimental cell populations.

Historically, research in spermatogonial biology has been hindered by a lack of validated approaches to identify and isolate pure populations of the various spermatogonial subsets for in-depth analysis. In particular, although a number of markers of the undifferentiated spermatogonial population have now been characterized, standardized methodology for assessing their specificity to the spermatogonial stem cell (SSC) and transit amplifying progenitor pools has been lacking. To date, SSC content within an undefined population of spermatogonia has been inferred using either lineage tracing or spermatogonial transplantation analyses which generate qualitative and quantitative data, respectively. Therefore, these techniques are not directly comparable, and are subject to variable interpretations as to a readout that is representative of a 'pure' SSC population. We propose standardization across the field for determining the SSC purity of a population via use of a limiting dilution transplantation assay that would eliminate subjectivity and help to minimize the generation of inconsistent data on 'SSC' populations. In the limiting dilution transplantation assay, a population of LacZ-expressing spermatogonia are selected based on a putative SSC marker, and a small, defined number of cells (i.e. 10 cells) are microinjected into the testis of a germ cell-deficient recipient mouse. Using colony counts and an estimated colonization efficiency of 5%; a quantitative value can be calculated that represents SSC purity in the starting population. The utilization of this technique would not only be useful to link functional relevance to novel markers that will be identified in the future, but also for providing validation of purity for marker-selected populations of spermatogonia that are commonly considered to be SSCs by many researchers in the field of spermatogenesis and stem cell biology.

Retinoic acid deficiency leads to an increase in spermatogonial stem number in the neonatal mouse testis, but excess retinoic acid results in no change.

The onset of spermatogenesis occurs in response to retinoic acid (RA), the active metabolite of vitamin A. However, whether RA plays any role during establishment of the spermatogonial stem cell (SSC) pool is unknown. Because designation of the SSC population and the onset of RA signaling in the testis that induces differentiation have similar timing, this study asked whether RA influenced SSC establishment. Whole mount immunofluorescence and flow cytometric analysis using the Id4-eGfp transgenic reporter mouse line revealed an enrichment for ID4-EGFP+ cells within the testis following inhibition of RA synthesis by WIN 18,446 treatment. Transplantation analyses confirmed a significant increase in the number of SSCs in testes from RA-deficient animals. Conversely, no difference in the ID4-EGFP+ population or change in SSC number were detected following exposure to an excess of RA. Collectively, reduced RA altered the number of SSCs present in the neonatal testis but precocious RA exposure in the neonatal testis did not, suggesting that RA deficiency causes a greater proportion of progenitor undifferentiated spermatogonia to retain their SSC state past the age when the pool is thought to be determined.

Glycolysis-Optimized Conditions Enhance Maintenance of Regenerative Integrity in Mouse Spermatogonial Stem Cells during Long-Term Culture.

The application of spermatogonial stem cell (SSC) transplantation for regenerating male fertility requires amplification of SSC number in vitro during which the integrity to re-establish spermatogenesis must be preserved. Conventional conditions supporting proliferation of SSCs from mouse pups have been the basis for developing methodology with adult human cells but are unrefined. We found that the integrity to regenerate spermatogenesis after transplantation declines with advancing time in primary cultures of pup SSCs and that the efficacy of deriving cultures from adult SSCs is limited with conventional conditions. To address these deficiencies, we optimized the culture environment to favor glycolysis as the primary bioenergetics process. In these conditions, regenerative integrity of pup and adult SSCs was significantly improved and the efficiency of establishing primary cultures was 100%. Collectively, these findings suggest that SSCs are primed for conditions favoring glycolytic activity, and matching culture environments to their bioenergetics is critical for maintaining functional integrity.

Recent advances for spermatogonial stem cell transplantation in livestock.

At the foundation of spermatogenesis are the actions of spermatogonial stem cells (SSCs), and a remarkable feature of these cells is the capacity to regenerate spermatogenesis following transplantation into testes of a recipient male that lacks endogenous germline. This ability could be exploited in livestock production as a breeding tool to enhance genetic gain. A key element to success is derivation of culture conditions that support proliferation of SSCs to provide sufficient numbers of cells for transfer into multiple recipient males. Using methodology devised for rodent cells as a foundation, advances in culturing cattle SSCs have occurred over the past few years and efforts are underway to extend this capability to pig cells. Another critical component to SSC transplantation is generation of males with germline ablation but intact somatic support cell function that can serve as surrogate sires for donor-derived spermatogenesis in a natural mating scheme. Recent advances in pigs using gene editing technologies have demonstrated that knockout of a key male germ cell-specific gene, namely NANOS2, leads to male-specific germline ablation but otherwise normal physiology, including intact seminiferous tubules. Together with recent advances in culturing spermatogonia of higher-order mammals, the now efficient means of producing germline-ablated recipient males have brought the application of SSC transplantation in livestock as a production tool closer to reality than ever before.

Transplantation as a Quantitative Assay to Study Mammalian Male Germline Stem Cells.

In mammals, the activities of spermatogonial stem cells (SSCs) provide the foundation for continual spermatogenesis throughout a male's reproductive lifetime. At present, the defining characteristics of SSCs and mechanisms controlling their fate decisions are not well understood. Transplantation is a definitive functional measure of stem cell capacity for male germ cells that can be used as an assay to provide an unequivocal quantification of the SSC content in an experimental cell population. Here, we discuss the procedure for mice and provide protocols for preparing donor germ cell suspensions from testes directly or primary cultures of spermatogonia for transplantation, enriching for SSCs, preparing recipient males, microinjection into recipient testes, and considerations for experimental design.

TSPAN8 Expression Distinguishes Spermatogonial Stem Cells in the Prepubertal Mouse Testis.

Precise separation of spermatogonial stem cells (SSCs) from progenitor spermatogonia that lack stem cell activity and are committed to differentiation remains a challenge. To distinguish between these spermatogonial subtypes, we identified genes that exhibited bimodal mRNA levels at the single-cell level among undifferentiated spermatogonia from Postnatal Day 6 mouse testes, including Tspan8, Epha2, and Pvr, each of which encode cell surface proteins useful for cell selection. Transplantation studies provided definitive evidence that a TSPAN8-high subpopulation is enriched for SSCs. RNA-seq analyses identified genes differentially expressed between TSPAN8-high and -low subpopulations that clustered into multiple biological pathways potentially involved in SSC renewal or differentiation, respectively. Methyl-seq analysis identified hypomethylated domains in the promoters of these genes in both subpopulations that colocalized with peaks of histone modifications defined by ChIP-seq analysis. Taken together, these results demonstrate functional heterogeneity among mouse undifferentiated spermatogonia and point to key biological characteristics that distinguish SSCs from progenitor spermatogonia.

Conditions for Long-Term Culture of Cattle Undifferentiated Spermatogonia.

Continual and robust spermatogenesis relies on the actions of an undifferentiated spermatogonial population that contains stem cells. A remarkable feature of spermatogonial stem cells (SSCs) is the capacity to regenerate spermatogenesis following isolation from a donor testis and transplantation into a permissive recipient testis. This capacity has enormous potential as a tool for enhancing the reproductive capacity of livestock, which can improve production efficiency. Because SSCs are a rare subset of the undifferentiated spermatogonial population, a period of in vitro amplification in number following isolation from donor testicular tissue is essential. Here, we describe methodology for isolation of a cell fraction from prepubertal bull testes that is enriched for undifferentiated spermatogonia and long-term maintenance of the cells in both the feeder cell coculture and the feeder-free format. To achieve this method, we derived bovine fetal fibroblasts (BFF) to serve as feeders for optimizing medium conditions that promote maintenance of bovine undifferentiated spermatogonia for at least 2 mo. In addition, we devised a feeder-free system with BFF-conditioned medium that sustained bovine undifferentiated spermatogonia for at least 1 mo in vitro. The methodologies described could be optimized to provide platforms for exponential expansion of bovine SSCs that will provide the numbers needed for transplantation into recipient testes.